An in vitro model system for measuring net alanine release from skeletal muscle has been developed. Using this system it has been shown that the synthesis of alanine can occur independent of transamination. Preliminary results suggest that the unknown reactions involve the sarcolemma and may proceed through neutral proteolysis. The purpose of this proposal is to design specific experiments to determine the nature of the hydrolytic process, to identify precursors, and to isolate the specific enzymes associated with this phenomenon. In addition, an in vitro system for measuring net flux of alanine from skeletal muscle will be designed. The correlated studies using these two systems will thus allow a detailed description of net amino acid flux from muscle. The nature of the specific substrates and the factors that regulate this flux will be identified using electrophoresis, chromatography, and isolation of labeled intermediates. The elucidation of the biochemical mechanism should contribute to our understanding of the regulation of protein turnover in muscle. Furthermore, its relationship to gluconeogenesis should help to explain the role of the peripheral tissue in maintaining nitrogen balance under a variety of physiological conditions.